Thermal injury represents a common form of trauma associated with significant mortality and morbidity. The end-organ effect of burn-induced systemic inflammation imposes a life-threatening problem even after successful initial resuscitation. One of the mechanisms underlying multiple organ failure is microvascular barrier dysfunction, a cellular process that has yet to be understood at the molecular level. The overall goal of this research project is to define the molecular mechanisms of microvascular leakage in thermal injury. Our initial investigation during the previous funding cycle has led to the development of unique experimental models that enable quantitative analyses of microvascular permeability. The experiments revealed a series of signaling and structural modifications in the endothelial barrier involving the contractile cytoskeleton and cell-cell adhesive interactions. As a continuing effort, we propose to extend this original investigation to a more in-depth analysis of the endothelial molecular response, with a practical view toward identifying new therapeutic targets for the effective treatment of burn edema. Our hypothesis states that fluid leak during severe burn occurs via the endothelial paracellular pathway caused by MLCK210-triggered actomyosin contraction and betacatenin serine phosphorylation-induced VE-cadherin dissociation. Selective inhibition of the contractile elements and stabilization of junctional complexes possess therapeutic potential as alternative means to attenuate the barrier injury. This hypothesis will be tested with a multifaceted approach that integrates novel cell biology techniques into physiological experiments. Through this study we wish to achieve the following specific aims: 1) to understand the molecular mechanisms of endothelial barrier dysfunction in thermal injury, and 2) to test the therapeutic effects of endothelial cytoskeleton-junction stabilizers in treating burn edema. Information gleaned from the study will significantly advance our understanding of microvascular pathobiology following thermal injury, with the potential to be translated to clinical practice for improved patient care.